Human infections with Entamoeba parasites are common;an estimated 500 million people are colonized with ameba resulting in 50 million cases of invasive amebiasis annually. The parasite's life cycle involves interconversion from a trophozoite to a cyst stage, a developmental pattern which is essential for pathogen propagation and disease causation. However, the factors regulating development of the parasite are poorly understood. The current model for studying Entamoeba stage conversion is E. invadens, a parasite which causes clinical disease in reptiles that is similar to human intestinal disease caused by E. histolytica. In E. invadens, both high level regulated encystation and excystation are possible. However, no genetic tools have been developed for E. invadens, significantly limiting the use of this system for characterizing the molecular basis of stage conversion. In this R03 application, we propose to develop genetic tools for E. invadens. In Aim 1, we will develop methods for stable transfection in E. invadens and approaches for overexpression and regulated expression of candidate genes. In Aim 2, we will develop methods for gene downregulation in E. invadens, including an approach that utilizes regulated downregulation. Successful development of genetic tools in E. invadens will allow us to dissect key aspects of the developmental biology of this parasite including identification of the pathways that trigger encystation. Tools and reagents generated by us will (i) be freely distributed, (ii) provide a valuable resource to the Entamoeba community, and (iii) increase the attractiveness of the model and thus the number of investigators studying this essential biological process in an important human pathogen. PUBLIC HEALTH RELEVANCE: Entamoeba histolytica is an important pathogen with an impact on human health on a global scale. Conversion between the two life cycle stages is crucial to disease transmission;however, this area of parasite biology is poorly understood. We are interested in understanding the molecular mechanisms that the parasite uses to convert between life cycle stages.